Genetic engineering, also called genetic modification, is the direct manipulation of an

organism's genome using biotechnology. New DNA may be inserted in the host genome by first

isolating and copying the genetic material of interest using molecular cloningmethods to generate a
DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host
organism. Genesmay be removed, or "knocked out", using a nuclease. Gene targeting is a different
technique that uses homologous recombination to change an endogenous gene, and can be used to
delete a gene, remove exons, add a gene, or introduce point mutations.

An organism that is generated through genetic engineering is considered to be a genetically

modified organism (GMO). The first GMOs were bacteria in 1973 and GM mice were generated in
1974. Insulin-producing bacteria were commercialized in 1982 and genetically modified food has
been sold since 1994. Glofish, the first GMO designed as a pet, was first sold in the United States
December in 2003.[1]

Genetic engineering techniques have been applied in numerous fields including research,
agriculture, industrial biotechnology, and medicine. Enzymes used in laundry detergent and
medicines such as insulin and human growth hormone are now manufactured in GM cells,
experimental GM cell lines and GM animals such as mice or zebrafish are being used for research
purposes, andgenetically modified crops have been commercialized.

Definition[edit]

Comparison of conventional plant breeding with transgenic and cisgenic genetic modification.
Genetic engineering alters the genetic make-up of an organism using techniques that
remove heritable material or that introduce DNA prepared outside the organism either directly into
the host or into a cell that is then fused or hybridized with the host.[4] This involves using recombinant
nucleic acid (DNA or RNA) techniques to form new combinations of heritable genetic material
followed by the incorporation of that material either indirectly through a vector system or directly
throughmicro-injection, macro-injection and micro-encapsulation techniques.

Genetic engineering does not normally include traditional animal and plant breeding, in vitro

fertilisation, induction ofpolyploidy, mutagenesis and cell fusion techniques that do not use
recombinant nucleic acids or a genetically modified organism in the process. [4] However the
European Commission has also defined genetic engineering broadly as including selective breeding
and other means of artificial selection. [5] Cloning and stem cell research, although not considered
genetic engineering,[6] are closely related and genetic engineering can be used within them.
[7]
 Synthetic biology is an emerging discipline that takes genetic engineering a step further by
introducing artificially synthesized genetic material from raw materials into an organism. [8]

If genetic material from another species is added to the host, the resulting organism is
called transgenic. If genetic material from the same species or a species that can naturally breed
with the host is used the resulting organism is called cisgenic.[9]Genetic engineering can also be used
to remove genetic material from the target organism, creating a gene knockoutorganism.[10] In Europe
genetic modification is synonymous with genetic engineering while within the United States of
America it can also refer to conventional breeding methods. [11][12] The Canadian regulatory system is
based on whether a product has novel features regardless of method of origin. In other words, a
product is regulated as genetically modified if it carries some trait not previously found in the species
whether it was generated using traditional breeding methods (e.g., selective breeding, cell
fusion,mutation breeding) or genetic engineering. [13][14][15] Within the scientific community, the
term genetic engineering is not commonly used; more specific terms such as transgenicare
preferred.

Genetically modified organisms[edit]

Main article: Genetically modified organism

Plants, animals or micro organisms that have changed through genetic engineering are termed
genetically modified organisms or GMOs.[16] Bacteria were the first organisms to be genetically
modified. Plasmid DNA containing new genes can be inserted into the bacterial cell and the bacteria
will then express those genes. These genes can code for medicines or enzymes that process food
and other substrates.[17][18] Plants have been modified for insect protection, herbicide resistance, virus
resistance, enhanced nutrition, tolerance to environmental pressures and the production of edible
vaccines.[19] Most commercialised GMO's are insect resistant and/or herbicide tolerant crop plants.
[20]
Genetically modified animals have been used for research, model animals and the production of
agricultural or pharmaceutical products. They include animals with genes knocked out, increased
susceptibility to disease, hormones for extra growth and the ability to express proteins in their milk. [21]

History[edit]
Main article: History of genetic engineering

Humans have altered the genomes of species for thousands of years through artificial selection and
more recently mutagenesis. Genetic engineering as the direct manipulation of DNA by humans
outside breeding and mutations has only existed since the 1970s. The term "genetic engineering"
was first coined by Jack Williamson in his science fiction novel Dragon's Island, published in 1951,
[22]
 one year before DNA's role in heredity was confirmed by Alfred Hershey and Martha Chase,[23] and
two years before James Watsonand Francis Crick showed that the DNA molecule has a double-helix
structure.

In 1974 Rudolf Jaenisch created the first GM animal.

In 1972 Paul Berg created the first recombinant DNA molecules by combining DNA from the monkey
virus SV40 with that of the lambda virus.[24] In 1973 Herbert Boyer and Stanley Cohen created the
first transgenic organism by inserting antibiotic resistance genes into theplasmid of an E.
coli bacterium. [25][26]
 A year later Rudolf Jaenisch created a transgenic mouse by introducing foreign
DNA into its embryo, making it the world’s first transgenic animal.[27] These achievements led to
concerns in the scientific community about potential risks from genetic engineering, which were first
discussed in depth at the Asilomar Conference in 1975. One of the main recommendations from this
meeting was that government oversight of recombinant DNA research should be established until
the technology was deemed safe.[28][29]
In 1976 Genentech, the first genetic engineering company, was founded by Herbert Boyer
and Robert Swanson and a year later the company produced a human protein (somatostatin)
in E.coli. Genentech announced the production of genetically engineered humaninsulin in 1978.[30] In
1980, the U.S. Supreme Court in the Diamond v. Chakrabarty case ruled that genetically altered life
could be patented.[31] The insulin produced by bacteria, branded humulin, was approved for release
by the Food and Drug Administration in 1982.[32]

In the 1970s graduate student Steven Lindow of the University of Wisconsin–Madison with D.C. Arny
and C. Upper found a bacterium he identified as P. syringae that played a role in ice nucleation and
in 1977, he discovered a mutant ice-minus strain. Later, he successfully created a recombinant ice-
minus strain.[33] In 1983, a biotech company, Advanced Genetic Sciences (AGS) applied for U.S.
government authorization to perform field tests with the ice-minus strain of P. syringae to protect
crops from frost, but environmental groups and protestors delayed the field tests for four years with
legal challenges.[34] In 1987, the ice-minus strain of P. syringae became the first genetically modified
organism (GMO) to be released into the environment [35] when a strawberry field and a potato field in
California were sprayed with it.[36] Both test fields were attacked by activist groups the night before
the tests occurred: "The world's first trial site attracted the world's first field trasher". [35]

The first field trials of genetically engineered plants occurred in France and the USA in 1986,
tobacco plants were engineered to be resistant to herbicides.[37] The People’s Republic of China was
the first country to commercialize transgenic plants, introducing a virus-resistant tobacco in 1992.
[38]
 In 1994 Calgene attained approval to commercially release the Flavr Savr tomato, a tomato
engineered to have a longer shelf life. [39] In 1994, the European Union approved tobacco engineered
to be resistant to the herbicidebromoxynil, making it the first genetically engineered crop
commercialized in Europe.[40] In 1995, Bt Potato was approved safe by the Environmental Protection
Agency, after having been approved by the FDA, making it the first pesticide producing crop to be
approved in the USA.[41] In 2009 11 transgenic crops were grown commercially in 25 countries, the
largest of which by area grown were the USA, Brazil, Argentina, India, Canada, China, Paraguay
and South Africa.[42]

In the late 1980s and early 1990s, guidance on assessing the safety of genetically engineered plants
and food emerged from organizations including the FAO and WHO.[43][44][45][46]

In 2010, scientists at the J. Craig Venter Institute, announced that they had created the first synthetic
bacterial genome. The researchers added the new genome to bacterial cells and selected for cells
that contained the new genome. To do this the cells undergoes a process called resolution, where
during bacterial cell division one new cell receives the original DNA genome of the bacteria, whilst
the other receives the new synthetic genome. When this cell replicates it uses the synthetic genome
as its template. The resulting bacterium the researchers developed, named Synthia, was the world's
first synthetic life form.[47][48]
Process[edit]
Main article: Techniques of genetic engineering

The first step is to choose and isolate the gene that will be inserted into the genetically modified
organism. As of 2012, most commercialised GM plants have genes transferred into them that
provide protection against insects or tolerance to herbicides. [49] The gene can be isolated
using restriction enzymes to cut DNA into fragments and gel electrophoresis to separate them out
according to length.[50] Polymerase chain reaction (PCR) can also be used to amplify up a gene
segment, which can then be isolated through gel electrophoresis. [51] If the chosen gene or the donor
organism's genome has been well studied it may be present in a genetic library. If the DNA
sequence is known, but no copies of the gene are available, it can be artificially synthesized.[52]

The gene to be inserted into the genetically modified organism must be combined with other genetic
elements in order for it to work properly. The gene can also be modified at this stage for better
expression or effectiveness. As well as the gene to be inserted most constructs contain
a promoter and terminator region as well as a selectable markergene. The promoter region
initiates transcription of the gene and can be used to control the location and level of gene
expression, while the terminator region ends transcription. The selectable marker, which in most
cases confers antibiotic resistance to the organism it is expressed in, is needed to determine which
cells are transformed with the new gene. The constructs are made using recombinant
DNA techniques, such as restriction digests, ligations and molecular cloning.[53] The manipulation of
the DNA generally occurs within a plasmid.

The most common form of genetic engineering involves inserting new genetic material randomly
within the host genome. [citation  Other techniques allow new genetic material to be inserted at a
needed]

specific location in the host genome or generate mutations at desired genomic loci capable

of knocking out endogenous genes. The technique ofgene targeting uses homologous
recombination to target desired changes to a specific endogenous gene. This tends to occur at a
relatively low frequency in plants and animals and generally requires the use of selectable markers.
The frequency of gene targeting can be greatly enhanced with the use of
engineered nucleases such as zinc finger nucleases, [54][55]
 engineered homing endonucleases, [56][57]
 or
nucleases created from TAL effectors. [58][59]
 In addition to enhancing gene targeting, engineered
nucleases can also be used to introduce mutations at endogenous genes that generate a gene
knockout.[60][61]

Transformation[edit]
Main article: Transformation (genetics)
A. tumefaciens attaching itself to a carrot cell

Only about 1% of bacteria are naturally capable of taking up foreign DNA. However, this ability can
be induced in other bacteria via stress (e.g.thermal or electric shock), thereby increasing the cell
membrane's permeability to DNA; up-taken DNA can either integrate with the genome or exist
as extrachromosomal DNA. DNA is generally inserted into animal cells using microinjection, where it
can be injected through the cell'snuclear envelope directly into the nucleus or through the use of viral
vectors.[62] In plants the DNA is generally inserted using Agrobacterium-mediated
recombination or biolistics. [63]

In Agrobacterium-mediated recombination, the plasmid construct contains T-DNA, DNA which is

responsible for insertion of the DNA into the host plants genome. This plasmid is transformed
into Agrobacterium containing no plasmids prior to infecting the plant cells. TheAgrobacterium will
then naturally insert the genetic material into the plant cells. [64] In biolistics transformation particles of
gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some
genetic material will enter the cells and transform them. This method can be used on plants that are
not susceptible to Agrobacterium infection and also allows transformation of plant plastids. Another
transformation method for plant and animal cells is electroporation. Electroporation involves
subjecting the plant or animal cell to an electric shock, which can make the cell membrane
permeable to plasmid DNA. In some cases the electroporated cells will incorporate the DNA into
their genome. Due to the damage caused to the cells and DNA the transformation efficiency of
biolistics and electroporation is lower than agrobacterial mediated transformation and microinjection.
[65]

As often only a single cell is transformed with genetic material the organism must be regenerated
from that single cell. As bacteria consist of a single cell and reproduce clonally regeneration is not
necessary. In plants this is accomplished through the use of tissue culture. Each plant species has
different requirements for successful regeneration through tissue culture. If successful an adult plant
is produced that contains the transgene in every cell. In animals it is necessary to ensure that the
inserted DNA is present in theembryonic stem cells. Selectable markers are used to easily
differentiate transformed from untransformed cells. These markers are usually present in the
transgenic organism, although a number of strategies have been developed that can remove the
selectable marker from the mature transgenic plant. [66] When the offspring is produced they can be
screened for the presence of the gene. All offspring from the first generation will be heterozygous for
the inserted gene and must be mated together to produce a homozygousanimal.

Further testing uses PCR, Southern hybridization, and DNA sequencing is conducted to confirm that

an organism contains the new gene. These tests can also confirm the chromosomal location and
copy number of the inserted gene. The presence of the gene does not guarantee it will
be expressed at appropriate levels in the target tissue so methods that look for and measure the
gene products (RNA and protein) are also used. These include northern hybridization,
quantitative RT-PCR, Western blot,immunofluorescence, ELISA and phenotypic analysis. For stable
transformation the gene should be passed to the offspring in a Mendelian inheritance pattern, so the
organism's offspring are also studied.

Genome editing[edit]
Main article: Genome editing

Genome editing is a type of genetic engineering in which DNA is inserted, replaced, or removed
from a genome using artificially engineered nucleases, or "molecular scissors." The nucleases
create specific double-stranded break (DSBs) at desired locations in the genome, and harness the
cell’s endogenous mechanisms to repair the induced break by natural processes of homologous
recombination (HR) and nonhomologous end-joining (NHEJ). There are currently four families of
engineered nucleases: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like
effector nucleases (TALENs), and CRISPRs.[67][68]

Applications[edit]
Genetic engineering has applications in medicine, research, industry and agriculture and can be
used on a wide range of plants, animals and micro organisms.

Medicine[edit]
In medicine, genetic engineering has been used to mass-produce insulin, human growth
hormones, follistim (for treating infertility), human albumin, monoclonal antibodies,antihemophilic
factors, vaccines and many other drugs.[69][70] Vaccination generally involves injecting weak, live, killed
or inactivated forms of viruses or their toxins into the person being immunized.[71] Genetically
engineered viruses are being developed that can still confer immunity, but lack
the infectious sequences.[72] Mouse hybridomas, cells fused together to create monoclonal
antibodies, have been humanised through genetic engineering to create human monoclonal
antibodies.[73] Genetic engineering has shown promise for treating certain forms of cancer. [74][75]
Genetic engineering is used to create animal models of human diseases. Genetically modified
mice are the most common genetically engineered animal model. [76] They have been used to study
and model cancer (the oncomouse), obesity, heart disease, diabetes, arthritis, substance abuse,
anxiety, aging and Parkinson disease. [77] Potential cures can be tested against these mouse models.
Also genetically modified pigs have been bred with the aim of increasing the success of  pig to
human organ transplantation.[78]

Gene therapy is the genetic engineering of humans by replacing defective human genes with
functional copies. This can occur in somatic tissue or germline tissue. If the gene is inserted into the
germline tissue it can be passed down to that person's descendants. [79][80] Gene therapy has been
successfully used to treat multiple diseases, including X-linked SCID,[81] chronic lymphocytic
leukemia (CLL),[82] and Parkinson's disease.[83] In 2012, Glybera became the first gene therapy
treatment to be approved for clinical use in either Europe or the United States after its endorsement
by the European Commission. [84][85] There are also ethical concerns should the technology be used
not just for treatment, but for enhancement, modification or alteration of a human beings'
appearance, adaptability, intelligence, character or behavior. [86] The distinction between cure and
enhancement can also be difficult to establish. [87] Transhumanists consider the enhancement of
humans desirable.

Research[edit]

Knockout mice
Human cells in which some proteins are fused with green fluorescent proteinto allow them to be visualised

Genetic engineering is an important tool for natural scientists. Genes and other genetic information
from a wide range of organisms are transformed into bacteria for storage and modification,
creating genetically modified bacteria in the process. Bacteria are cheap, easy to grow, clonal,
multiply quickly, relatively easy to transform and can be stored at -80 °C almost indefinitely. Once a
gene is isolated it can be stored inside the bacteria providing an unlimited supply for research.

Organisms are genetically engineered to discover the functions of certain genes. This could be the
effect on the phenotype of the organism, where the gene is expressed or what other genes it
interacts with. These experiments generally involve loss of function, gain of function, tracking and
expression.

 Loss of function experiments, such as in a gene knockout experiment, in which an

organism is engineered to lack the activity of one or more genes. A knockout experiment
involves the creation and manipulation of a DNA construct in vitro, which, in a simple knockout,
consists of a copy of the desired gene, which has been altered such that it is non-
functional. Embryonic stem cells incorporate the altered gene, which replaces the already
present functional copy. These stem cells are injected into blastocysts, which are implanted into
surrogate mothers. This allows the experimenter to analyze the defects caused by
this mutation and thereby determine the role of particular genes. It is used especially frequently
in developmental biology. Another method, useful in organisms such as Drosophila(fruit fly), is to
induce mutations in a large population and then screen the progeny for the desired mutation. A
similar process can be used in both plants and prokaryotes.
 Gain of function experiments, the logical counterpart of knockouts. These are sometimes
performed in conjunction with knockout experiments to more finely establish the function of the
desired gene. The process is much the same as that in knockout engineering, except that the
construct is designed to increase the function of the gene, usually by providing extra copies of
the gene or inducing synthesis of the protein more frequently.

 Tracking experiments, which seek to gain information about the localization and interaction
of the desired protein. One way to do this is to replace the wild-type gene with a 'fusion' gene,
which is a juxtaposition of the wild-type gene with a reporting element such asgreen fluorescent
protein (GFP) that will allow easy visualization of the products of the genetic modification. While
this is a useful technique, the manipulation can destroy the function of the gene, creating
secondary effects and possibly calling into question the results of the experiment. More
sophisticated techniques are now in development that can track protein products without
mitigating their function, such as the addition of small sequences that will serve as binding motifs
to monoclonal antibodies.
 Expression studies aim to discover where and when specific proteins are produced. In
these experiments, the DNA sequence before the DNA that codes for a protein, known as a
gene's promoter, is reintroduced into an organism with the protein coding region replaced by a
reporter gene such as GFP or an enzyme that catalyzes the production of a dye. Thus the time
and place where a particular protein is produced can be observed. Expression studies can be
taken a step further by altering the promoter to find which pieces are crucial for the proper
expression of the gene and are actually bound by transcription factor proteins; this process is
known as promoter bashing.

Industrial[edit]
Using genetic engineering techniques one can transform microorganisms such as bacteria or yeast,
or transform cells from multicellular organisms such as insects or mammals, with a gene coding for a
useful protein, such as an enzyme, so that the transformed organism will overexpress the desired
protein. One can manufacture mass quantities of the protein by growing the transformed organism
in bioreactor equipment using techniques of industrial fermentation, and then purifying the protein.
[88]
 Some genes do not work well in bacteria, so yeast, insect cells, or mammalians cells, each
a eukaryote, can also be used.[89] These techniques are used to produce medicines such
as insulin, human growth hormone, and vaccines, supplements such as tryptophan, aid in the
production of food (chymosin in cheese making) and fuels.[90] Other applications involving genetically
engineered bacteria being investigated involve making the bacteria perform tasks outside their
natural cycle, such as making biofuels,[91] cleaning up oil spills, carbon and other toxic waste [92] and
detecting arsenic in drinking water.[93]

Experimental, lab scale industrial applications[edit]

In materials science, a genetically modified virus has been used in an academic lab as a scaffold for
assembling a more environmentally friendly lithium-ion battery. [94][95]

Bacteria have been engineered to function as sensors by expressing a fluorescent protein under
certain environmental conditions.[96]

Agriculture[edit]
Main article: Genetically modified crops
Bt-toxins present in peanut leaves (bottom image) protect it from extensive damage caused by European corn
borer larvae (top image).[97]

One of the best-known and controversial applications of genetic engineering is the creation and use
of genetically modified crops orgenetically modified organisms, such as genetically modified fish,
which are used to produce genetically modified food and materials with diverse uses. There are four
main goals in generating genetically modified crops.[98]

One goal, and the first to be realized commercially, is to provide protection from environmental
threats, such as cold (in the case of Ice-minus bacteria), or pathogens, such as insects or viruses,
and/or resistance to herbicides. There are also fungal and virus resistant crops developed or in
development.[99][100] They have been developed to make the insect and weed management of crops
easier and can indirectly increase crop yield.[101]

Another goal in generating GMOs is to modify the quality of produce by, for instance, increasing the
nutritional value or providing more industrially useful qualities or quantities. [102] The Amflora potato, for
example, produces a more industrially useful blend of starches. Cows have been engineered to
produce more protein in their milk to facilitate cheese production. [103] Soybeans and canola have been
genetically modified to produce more healthy oils.[104][105]

Another goal consists of driving the GMO to produce materials that it does not normally make. One
example is "pharming", which uses crops as bioreactors to produce vaccines, drug intermediates, or
drug themselves; the useful product is purified from the harvest and then used in the standard
pharmaceutical production process.[106] Cows and goats have been engineered to express drugs and
other proteins in their milk, and in 2009 the FDA approved a drug produced in goat milk. [107][108]

Another goal in generating GMOs, is to directly improve yield by accelerating growth, or making the
organism more hardy (for plants, by improving salt, cold or drought tolerance). [102] Some agriculturally
important animals have been genetically modified with growth hormones to increase their size. [109]

The genetic engineering of agricultural crops can increase the growth rates and resistance to
different diseases caused by pathogens andparasites.[110] This is beneficial as it can greatly increase
the production of food sources with the usage of fewer resources that would be required to host the
world's growing populations. These modified crops would also reduce the usage of chemicals, such
as fertilizers andpesticides, and therefore decrease the severity and frequency of the damages
produced by these chemical pollution.[110][111]

Ethical and safety concerns have been raised around the use of genetically modified food. [112] A
major safety concern relates to the human health implications of eating genetically modified food, in
particular whether toxic or allergic reactions could occur. [113] Gene flow into related non-transgenic
crops, off target effects on beneficial organismsand the impact on biodiversity are important
environmental issues.[114] Ethical concerns involve religious issues, corporate control of the food
supply, intellectual property rights and the level of labeling needed on genetically modified products.

BioArt and entertainment[edit]

Genetic engineering is also being used to create BioArt.[115] Some bacteria have been genetically
engineered to create black and white photographs. [116]

Genetic engineering has also been used to create novelty items such as lavender-
colored carnations,[117] blue roses,[118] and glowing fish.[119][120]

Regulation[edit]
Main articles: Regulation of genetic engineering and Regulation of the release of genetically
modified organisms

The regulation of genetic engineering concerns the approaches taken by governments to assess
and manage the risks associated with the development and release of genetically modified crops.
There are differences in the regulation of GM crops between countries, with some of the most
marked differences occurring between the USA and Europe. Regulation varies in a given country
depending on the intended use of the products of the genetic engineering. For example, a crop not
intended for food use is generally not reviewed by authorities responsible for food safety.
CONTROVERSIES

Critics have objected to use of genetic engineering per se on several grounds, including ethical
concerns, ecological concerns, and economic concerns raised by the fact GM techniques and GM
organisms are subject to intellectual property law. GMOs also are involved in controversies over GM
food with respect to whether food produced from GM crops is safe, whether it should be labeled, and
whether GM crops are needed to address the world's food needs. See the genetically modified food
controversies article for discussion of issues about GM crops and GM food. These controversies
have led to litigation, international trade disputes, and protests, and to restrictive regulation of
commercial products in some countries.

Processes involving recombinant DNA technology

Using techniques such as restriction enzymes, ligation, polymerase chain reactions and
gel electrophoresis, scientists can now use recombinant DNA technology for processes
such as gene cloning  , producing transgenic organisms and DNA profiling or 'finger
printing'.

Transgenic organisms (TGOs)

Transgenic organisms are individuals with one or more genes that have been artificially
introduced from another species.

Recombinant DNA technology is used to produce transgenic organisms that have been
genetically modified for a specific purpose. The process has been applied to plants,
animals and bacteria.

Through this technology transgenic organisms have been produced for the production
of specific proteins, such as the hormone insulin, as well as vaccines, anticoagulants
and interferon.

Producing recombinant DNA

The production of recombinant DNA as a result of the action of the restriction enzyme
EcoRI and ligation is shown in the following diagram.
The first recombinant DNA experiments used bacteria.

Bacteria contain a large strand of chromosomal DNA which holds the main genetic
information of the bacteria as well as several smaller rings of double-stranded DNA
called plasmids.

Plasmids contain genes that provide protection against antibodies from other bacteria.

Plasmids are used as vectors   in producing recombinant DNA as explained below.
 Steps in producing recombinant DNA

1. The required gene is cut from a DNA molecule using a restriction enzyme.
2. A bacterial plasmid is isolated and cut with the same restriction enzyme. This
ensures cut ends are complementary (same base sequence) to the ends of the
required gene.
3. The required gene is joined to the plasmid using the enzyme DNA ligase in a
process called ligation. 
4. The resulting recombinant plasmid is returned to the bacterial cell. 
5. The bacteria reproduce and the required gene is cloned.

Recombinant DNA has also been produced in other organisms including certain fungi,
plants and animals. Viruses have also been used as vectors of recombinant DNA.

Yeast cells
Yeast cells, like bacteria, contain plasmids so the technique is the same as for bacteria.

Plant cells
Before genes can be introduced into plant cells the cell wall is removed by enzyme
action. This leaves the cell protoplast. Temporary holes are produced in the cell
membrane using an electrical pulse. The required genes can now enter the cell via the
holes, eg the transfer of disease-resistant genes from one plant species to another plant
species susceptable to the same disease.
Mammal (animal) cells
The required gene is injected, using a micropipette, into the nucleus of the animal cell,
eg in a trial experiment the gene for the rat growth hormone was injected into the
fertilised egg of a mouse to produce mice that are twice the weight and size of normal
mice. These 'giant mice' were the first transgenic animals to be produced.

Viruses
The required gene is combined with the virual DNA using DNA ligase to produce
recombinant DNA. When the virus 'infects' a host cell the required gene is introduced
into the organism.

 Expression studies aim to discover where and when specific proteins are produced. In
these experiments, the DNA sequence before the DNA that codes for a protein, known as a
gene's promoter, is reintroduced into an organism with the protein coding region replaced by a
reporter gene such as GFP or an enzyme that catalyzes the production of a dye. Thus the time
and place where a particular protein is produced can be observed. Expression studies can be
taken a step further by altering the promoter to find which pieces are crucial for the proper
expression of the gene and are actually bound by transcription factor proteins; this process is
known as promoter bashing.

• Transformation- process of introducing free DNA into bacteria

Competent cell- a cell that is capable of taking up DNA.

Electroporation- The use of an electric shock to momentarily open or disrupt cell walls.

Conjugation- the contact of bacteria that involves the exchange of DNA with a
mating tube.

Transformed cell- cell with new DNA

Marker gene- a gene that identifies which organisms have been successfully
transformed
Process of Genetic Engineering

1. Isolation:
• Removal of human DNA (containing target gene).
• Removal of plasmid (bacterial DNA) from bacterium.
• Cutting:
• Both human DNA and plasmid DNA are cut with the same restriction
enzyme.
• Normally plasmid has only one restriction site while human DNA will have
many restriction sites.

Insertion:

• means that target gene is placed into the DNA of the plasmid or cloning
vector.
• cut plasmids are mixed with human DNA sections allowing the cut ends to
combine.
Bacteria Boost Sunscreen
AUG 12, 2013 02:28 PM ET // BY NIC HALVERSON

A sunlight-absorbing compound that exists naturally in microalgae has been discovered at

the bottom of a Norwegian lake, and it could be used to create a more powerful
sunscreen.

SINTEF, a research institute in northern Norway, has been dredging the Trondheim Fjord
for several years and cataloging microorganisms that absorb sunlight. From one such
organism, researchers have extracted a substance called sarcinaxanthin, which possesses
a pigment that can absorb long wavelengths of UV radiation.
Freeze-Dry Tech Preserves Animal
Sperm
AUG 28, 2013 01:25 PM ET // BY AFP

The technology makes it possible to store sperm at room temperature for short periods, meaning it
would remain safe in the event of a power failure.

Japanese scientists have launched a sperm bank for endangered animals that uses
freeze-drying technology they hope could one day help humans recreate animal
populations on other planets, the chief researcher said Wednesday.
The team at Kyoto University's Institute of Laboratory Animals Graduate School of
Medicine successfully preserved sperm taken from two endangered primates and a type of
giraffe, associate professor Takehito Kaneko said.

Sperm Donors May Father 'Dozens' of Children

  PLAY VIDEO

Sperm Is The New Superfood

Yeah, it's gross, but our bodily fluids actually have a lot of nutrients in them! Anthony unveils several common
bodily excretions that are actually good for us.
ISTOCKPHOTO

They mixed the sperm with special preservation liquid and freeze-dried it in a way that
allows them to store it at just 4 degrees Celsius (39 Fahrenheit), Kaneko said.

The temperature is much higher -- and less energy intensive -- than conventional ways of
storing sperm.

A Bio-Patch Regrows Bone Inside the

Body
NOV 8, 2013 09:23 AM ET // BY GEORGE DVORSKY, IO9.COM
 Researchers from the University of Iowa have developed a remarkable new
procedure for regenerating missing or damaged bone. It's called a "bio patch" -- and it
works by sending bone-producing instructions directly into cells using microscopic
particles embedded with DNA.

In experiments, the gene-encoding patch has already regrown bone fully enough to cover
skull wounds in test animals. It has also stimulated new growth in human bone marrow
stromal cells. Eventually, the patch could be used to repair birth defects involving missing
bone around the head or face. It could also help dentists rebuild bone in areas which
provides a concrete-like foundation for implants.

BIOTECHNOLOGY

Mutated Virus Helps Build a Better Battery

NOV 14, 2013 02:25 PM ET // BY IO9.COM

A benign bacteria virus serves as a template in water to grow manganese oxide nanowires that, when
combined with palladium, increases the energy potential of a lithium battery.

By unleashing a genetically modified virus onto microscopic electrode wires, researchers

from MIT have shown that the performance of lithium-air batteries can be significantly
improved -- a remarkable breakthrough that could revolutionize the way our electric
devices are powered.

Indeed, lithium-air batteries have generated considerable buzz over the years because of
the way they can increase power without having to increase weight, an attribute that
could lead to electric cars with much greater driving range. But engineers have struggled
to to create the durable materials required for the batteries' electrodes, and increase the
number of charging-cycles the batteries can withstand.
Living Drug' Kills Leukemia
FEB 20, 2014 02:05 PM ET // BY AFP

A colored scanning electron micrograph shows red blood cells (orange) and B lymphocyte white blood
cells (white) from a patient with leukemia.

A new approach to killing cancer cells that uses a patient's own immune system has
beaten back leukemia in 88 percent of adults, US researchers said.

The report by scientists in New York offers more good news for the burgeoning field of
cancer immunotherapy, which uses what some describe as a "living drug" that was hailed
by Science magazine as the breakthrough of 2013.
 Glow-in-the-Dark Plants Go on Sale
MAR 27, 2014 11:50 AM ET // BY LINA ZELDOVICH, FOXNEWS.COM/SCIENCE

Fans of Avatar’s Pandora forests rejoice: glow-in-the-dark plants are coming to your
house, says Anthony Evans, the CEO of a synthetic biology startup that created the
bioluminescent flora. Glowing trees will take awhile to make, but you can already
preorder seeds of a glow-in-the-dark Arabidopsis, a little flowering plant of a mustard
family. According to the original Kickstarter campaign last year,Glowingplant.com would
start shipping the luminous Arabidopsis seeds to customers this April, but postponed the
release date till fall.

Evans said that the delay is because the company raised more money than expected and
could afford more work on tweaking their product to high shine. “We asked our backers a
few months ago whether they wanted us to ship on time or to use the rest of the funds to
improve the luminosity,” Evans told Fox News. “The overwhelming advice was to
improve.”
Powerful Gene-Mutating Tech Needs
More Debate
SEP 30, 2014 09:35 AM ET // BY HILLARY VAUGHN, FOXNEWS

A powerful new technology could be used to manipulate nature by changing a species

gene pool through reproduction, and it has scientists proceeding with caution.

The technology is called “gene drive” by Harvard scientists who say it allows them to
“edit” genes in wild organisms. Dr. Kenneth Oye, a professor at Massachusetts Institute of
Technology, says gene drives are a game changer. “Gene drives cheat, they play a game,"
he explained. "They bias inheritance so the odds of the gene being passed on are raised
substantially.”

For scientists, the possibilities of the technology are great. Gene drive lets researchers
permanently block mosquitoes’ ability to spread malaria, for example. It can also be used
to alter ticks, reducing the spread of Lyme disease.
“But its not just malaria -- any disease spread by mosquitos we can combat in this way,"
said Oye. "So that means dengue, that means yellow fever, that means, potentially closer
to home, West Nile Virus.”

Because the technology has the potential to alter entire populations on a global scale
scientists are proceeding with caution. The Harvard group is calling for public debate on
the wisdom and safety of the technology before moving forward.

“Ethically I think it's an open and shut case. A slam dunk. That if you’re going to be taken
actions that potentially affect the world, the world has a little voice in this,” said Oye.

For scientists the extent of the risk -- and consequences associated with the technology
are unknown. Scientists say that if this tech goes awry, it could mean accidental
extinction for entire species, or unpredictable gene re-mutations spreading cross-species.

Kevin Esvelt, lead scientist in the research group, says the technology needs to be used
wisely. “Yes, we should be concerned. We should come together and discuss this because
it has the potential to do a great deal of good, but it could also do harm if we use it
unwisely.”

But scientists say they have a plan B if something goes wrong. “We can release a second
drive that will undo that alteration and restore it pretty close to exactly the same
sequence as it was originally,” said Esvelt. But he warns the fix isn’t absolute. “Now that
doesn’t mean that it’s going to reverse all the potential ecological effects of that original
change. But it does mean that we can take precautions.”

The technology has even made waves internationally. A United Nations meeting of
experts on biological weapons convened to review the benefits and potential implications
of gene drive. Dr. Oye, who presented the tech to international delegates gathered in
Geneva, said the tech especially piqued the interest of delegations from Malaria-inflicted
regions. “The reaction at Geneva from the biosecurity experts, these are the hard core
folks, was very, very positive,” he said.

Developers of the tech say it's still in the developmental stages, but scientist George
Church says field trials could be just around the corner. “This technology could go very
quickly," he said. "We’re talking about something where we could be doing key tests on
four different organisms within the next year.”